Isolation and Properties of Lysine-producing Mutants with Feedbackresistant Aspartokinase Derived from a Brevibacterium flavum Strain with Citrate Synthase- and Pyruvate Kinase-defects and Feedback-resistant Phosphoenolpyruvate Carboxylase

Abstract

Although the growth of Brevibacterium flavum KH-21 with CS^L, PK^-, and PC^R was resistant to a lysine analogue, AEC, plus Thr at a concentration of 1 to 3 g/1, used conventionally for isolation of AK^R mutants, it was inhibited by higher concentrations of them, and recovered partially upon the addition of Lys, DAP and Met. The growth level recovered was equal to that in the presence of Thr alone. Several lysine-producing mutants with AK^R were isolated as those resistant to these high concentrations of AEC plus Thr. The representative strain, AH-198, produced 51 g/1 of Lys•HCl at maximum, when cultured for 72 hr in a medium containing 100 g/1 of glucose, while the parent strain KH-21 produced only 2 g/1. The productivity of strain AH-198 was the same as or a little higher than that of the HD^- type lysine-producing strain No. 22 with CS^L, PK^-, and PC^R. I_0.5, the concentration of Lys and Thr inhibiting 50% of the AK activity was 40 mM, 267-fold that of the parent enzyme. The Km for Asp and the optimum concentration of (NH₄)₂SO₄ for the apparent V_max were also increased. In addition, the inhibition by Thr or Lys alone disappeared in the mutant enzyme.